Pro-apoptotic Response Research Articles

Lipogenic transcription factor ChREBP mediates fructose-induced metabolic adaptations to prevent hepatotoxicity.

Epidemiologic and animal studies implicate overconsumption of fructose in the development of nonalcoholic fatty liver disease, but the molecular mechanisms underlying fructose-induced chronic liver diseases remain largely unknown. Here, we have presented evidence supporting the essential function of the lipogenic transcription factor carbohydrate response element-binding protein (ChREBP) in mediating adaptive responses to fructose and protecting against fructose-induced hepatotoxicity. In WT mice, a high-fructose diet (HFrD) activated hepatic lipogenesis in a ChREBP-dependent manner; however, in Chrebp-KO mice, a HFrD induced steatohepatitis. In Chrebp-KO mouse livers, a HFrD reduced levels of molecular chaperones and activated the C/EBP homologous protein-dependent (CHOP-dependent) unfolded protein response, whereas administration of a chemical chaperone or Chop shRNA rescued liver injury. Elevated expression levels of cholesterol biosynthesis genes in HFrD-fed Chrebp-KO livers were paralleled by an increased nuclear abundance of sterol regulatory element-binding protein 2 (SREBP2). Atorvastatin-mediated inhibition of hepatic cholesterol biosynthesis or depletion of hepatic Srebp2 reversed fructose-induced liver injury in Chrebp-KO mice. Mechanistically, we determined that ChREBP binds to nuclear SREBP2 to promote its ubiquitination and destabilization in cultured cells. Therefore, our findings demonstrate that ChREBP provides hepatoprotection against a HFrD by preventing overactivation of cholesterol biosynthesis and the subsequent CHOP-mediated, proapoptotic unfolded protein response. Our findings also identified a role for ChREBP in regulating SREBP2-dependent cholesterol metabolism.

The ubiquitin ligase LIN41/TRIM71 targets p53 to antagonize cell death and differentiation pathways during stem cell differentiation

Rapidity and specificity are characteristic features of proteolysis mediated by the ubiquitin-proteasome system. Therefore, the UPS is ideally suited for the remodeling of the embryonic stem cell proteome during the transition from pluripotent to differentiated states and its inverse, the generation of inducible pluripotent stem cells. The Trim-NHL family member LIN41 is among the first E3 ubiquitin ligases to be linked to stem cell pluripotency and reprogramming. Initially discovered in C. elegans as a downstream target of the let-7 miRNA, LIN41 is now recognized as a critical regulator of stem cell fates as well as the timing of neurogenesis. Despite being indispensable for embryonic development and neural tube closure in mice, the underlying mechanisms for LIN41 function in these processes are poorly understood. To better understand the specific contributions of the E3 ligase activity for the stem cell functions of LIN41, we characterized global changes in ubiquitin or ubiquitin-like modifications using Lin41-inducible mouse embryonic stem cells. The tumor suppressor protein p53 was among the five most strongly affected proteins in cells undergoing neural differentiation in response to LIN41 induction. We show that LIN41 interacts with p53, controls its abundance by ubiquitination and antagonizes p53-dependent pro-apoptotic and pro-differentiation responses. In vivo, the lack of LIN41 is associated with upregulation of Grhl3 and widespread caspase-3 activation, two downstream effectors of p53 with essential roles in neural tube closure. As Lin41-deficient mice display neural tube closure defects, we conclude that LIN41 is critical for the regulation of p53 functions in cell fate specification and survival during early brain development.

Preexposure of MCF-7 breast cancer cell line to dexamethasone alters the cytotoxic effect of paclitaxel but not 5-fluorouracil or epirubicin chemotherapy.

PurposeGlucocorticoids (GCs) are often administered prior to any chemotherapeutics to prevent the secondary effects of anticancer agents. Glucocorticoid receptors (GRs) are expressed in several types of cancer cells, particularly in several histological types of breast cancer. Activation of GRs is not associated with any specific cellular response. Both proapoptotic and antiapoptotic responses have been observed, depending on the study or the type of breast cancer cells. Therefore, it is of relevance to investigate the possible modulation of apoptotic effect of chemotherapeutic agents when cancerous cells have previously been exposed to GCs.MethodsIn vitro cell growth was assayed by counting MCF-7 cells upon exposure to epirubicin (25 nM), 5-fluorouracil (5-FU) (15 µM), and paclitaxel (15 nM), either with or without prior exposure to the GC dexamethasone (Dex) (100 nM).ResultsFollowing preexposure to Dex, the antiapoptotic activity of paclitaxel was significantly reduced by 8.5% (p<0.05), but the activities of epirubicin and 5-FU remained unaltered.ConclusionIn light of the finding that the response of MCF-7 cells pretreated with Dex was significantly reduced, we recommend that the function of GCs should be defined more precisely if they are to be used in conjunction with chemotherapy.

Abstract P3-03-02: Inhibition of NOS promotes ER stress response and augments docetaxel-mediated apoptosis in TNBC

Abstract Introduction: Chemoresistance in triple negative breast cancer (TNBC) is related to an activation of a survival response orchestrated by endoplasmic reticulum (ER) stress. We hypothesize that attenuation of nitric oxide (NO) signaling pathway can overcome treatment resistance, preventing relapse, ultimately improving survival of TNBC patients. Here, we aimed to investigate the effects of pharmacological iNOS (inducible nitric oxide synthase) inhibition by L-NMMA on docetaxel-meditated ER stress response and to determine whether the therapeutic NOS inhibition may improve chemotherapy-based response. Methods: BT-549, SUM-149, MDA-MB-436, and MDA-MD-468 TNBC cell lines were treated with docetaxel (D; 5 nm)/ L-NMMA (L; 4mM)/ amlodipine (A; 5 µm) daily for 48 and 72 hours. Cell death and proliferation were assayed by Annexin V and ATP quantification, respectively. Western Blot (WB) was used to measure ER stress markers. In vivo regimen treatment followed three 2-weekscycles of D (20 mg/kg intraperitoneal [IP] on day 1) and L (200 mg/kg oral gavage on day 2-6); A (10 mg/kg IP on day 2-6) A was administered together with L to counteract the well-known effects of L on blood pressure (hypertension). TNBC Patient derived xenograft (PDX) models #2147, #5998, #3107 and #4664 were transplanted into the mammary fat pad of SCID Beige mice. PDX #2147 received either, single drug (vehicle, L, A, D), double (L+A, D+L, D+A), or triple drug combination (L+A+D). Models #4664, #3107 and #5998 received only vehicle, D or D+ L+A. Mice weight and tumor volumes were recorded twice weekly. D concentration was measured by mass spectrometry. Results: Studies on SUM-159 cell line showed that, when compared to the docetaxel-treated group, D+L+A increased cell death significantly, as indicated by a rise in annexin V/propidium iodide-positive cells. Increase in cell death by D+L+A was further demonstrated by accumulation of mitochondrial cleaved BAX. The enhanced apoptotic effects of D+L+A in MDA MD 468, BT 549 and MDA MD TNBC cell lines were confirmed by a decrease in ATP levels compared to D alone. WB revealed a survival stress response activated by docetaxel. When it was coupled with NOS inhibition, ER stress response showed higher expression of ATF4 and CHOP, triggering a proapoptotic response by pASK1/JNK pathway and cleaved caspases (CC3 and CC9). PDX #2147 showed that L, A and L+A treatment groups had similar tumor volume growth as the untreated group. However, combination therapy, D+L+A, significantly reduced the tumor volume and increased survival proportions compared with vehicle and docetaxel. Combination therapy also dramatically reduced tumor size on TNBC #4664 and #3107, and significantly improved response on #5998 compared with docetaxel alone. Intratumoral docetaxel concentration was 5.3-fold higher in mice receiving D+L+A than in those receiving docetaxel alone (#5998). In both groups, docetaxel was not detected in the plasma one week after injection. Conclusion: The present data suggest that iNOS may be a critical target for docetaxel resistance in TNBC. iNOS inhibition enhanced chemotherapy response in TNBC PDX models indicating that addition of iNOS inhibitor may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. Citation Format: Davila-Gonzalez D, Choi DS, Kuhn J, Granados SM, Rosato RR, Dave B, Chang JC. Inhibition of NOS promotes ER stress response and augments docetaxel-mediated apoptosis in TNBC [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-02.

Correction: Hepatitis C Virus Sensing by Human Trophoblasts Induces Innate Immune Responses and Recruitment of Maternal NK Cells: Potential Implications for Limiting Vertical Transmission.

Hepatitis C virus (HCV) is the world's most common blood-borne viral infection for which there is no vaccine. The rates of vertical transmission range between 3 and 6% with odds 90% higher in the presence of HIV coinfection. Prevention of vertical transmission is not possible because of lack of an approved therapy for use in pregnancy or an effective vaccine. Recently, HCV has been identified as an independent risk factor for preterm delivery, perinatal mortality, and other complications. In this study, we characterized the immune responses that contribute to the control of viral infection at the maternal-fetal interface (MFI) in the early gestational stages. In this study, we show that primary human trophoblast cells and an extravillous trophoblast cell line (HTR8), from first and second trimester of pregnancy, express receptors relevant for HCV binding/entry and are permissive for HCV uptake. We found that HCV-RNA sensing by human trophoblast cells induces robust upregulation of type I/III IFNs and secretion of multiple chemokines that elicit recruitment and activation of decidual NK cells. Furthermore, we observed that HCV-RNA transfection induces a proapoptotic response within HTR8 that could affect the morphology of the placenta. To our knowledge, for the first time, we demonstrate that HCV-RNA sensing by human trophoblast cells elicits a strong antiviral response that alters the recruitment and activation of innate immune cells at the MFI. This work provides a paradigm shift in our understanding of HCV-specific immunity at the MFI as well as novel insights into mechanisms that limit vertical transmission but may paradoxically lead to virus-related pregnancy complications.

Cytokine-Induced Glucocorticoid Resistance from Eosinophil Activation: Protein Phosphatase 5 Modulation of Glucocorticoid Receptor Phosphorylation and Signaling.

The mechanisms contributing to persistent eosinophil activation and poor eosinopenic response to glucocorticoids in severe asthma are poorly defined. We examined the effect of cytokines typically overexpressed in the asthmatic airways on glucocorticoid signaling in in vitro activated eosinophils. An annexin V assay used to measure eosinophil apoptosis showed that cytokine combinations of IL-2 plus IL-4 as well as TNF-α plus IFN-γ, or IL-3, GM-CSF, and IL-5 alone significantly diminished the proapoptotic response to dexamethasone. We found that IL-2 plus IL-4 resulted in impaired phosphorylation and function of the nuclear glucocorticoid receptor (GCR). Proteomic analysis of steroid sensitive and resistant eosinophils identified several differentially expressed proteins, namely protein phosphatase 5 (PP5), formyl peptide receptor 2, and annexin 1. Furthermore, increased phosphatase activity of PP5 correlated with impaired phosphorylation of the GCR. Importantly, suppression of PP5 expression with small interfering RNA restored proper phosphorylation and the proapoptotic function of the GCR. We also examined the effect of lipoxin A4 on PP5 activation by IL-2 plus IL-4. Similar to PP5 small interfering RNA inhibition, pretreatment of eosinophils with lipoxin A4 restored GCR phosphorylation and the proaptoptotic function of GCs. Taken together, our results showed 1) a critical role for PP5 in cytokine-induced resistance to GC-mediated eosinophil death, 2) supported the dependence of GCR phosphorylation on PP5 activity, and 3) revealed that PP5 is a target of the lipoxin A4-induced pathway countering cytokine-induced resistance to GCs in eosinophils.

Reduced risk of apoptosis: mechanisms of stress responses.

Apoptosis signaling pathways are integrated into a wider network of interconnected apoptotic and anti-apoptotic pathways that regulate a broad range of cell responses from cell death to growth, development and stress responses. An important trigger for anti- or pro-apoptotic cell responses are different forms of stress including hypoxia, energy deprivation, DNA damage or inflammation. Stress duration and intensity determine whether the cell's response will be improved cell survival, due to stress adaptation, or cell death by apoptosis, necrosis or autophagy. Although the interplay between enhanced stress tolerance and modulation of apoptosis triggering is not yet fully understood, there is a substantial body of experimental evidence demonstrating that apoptosis and anti-apoptosis signaling pathways can be manipulated to trigger or delay apoptosis in vitro or in vivo. Anti-apoptotic strategies cover a broad range of approaches. These interventions include mediators that prevent apoptosis (trophic factors and cytokines), apoptosis inhibition (caspase inhibition, stimulation of anti-apoptotic or inhibition of pro-apoptotic proteins and elimination of apoptotic stimulus), adaptive stress responses (induction of maintenance and repair, caspase inactivation) and cell-cell interactions (blocking engulfment and modified micro environment). There is a consensus that preclinical efficacy and safety evaluations of anti-apoptotic strategies should be performed with protocols that simulate as closely as possible the effects of aging, gender, risk factors, comorbidities and co-medications.

The intrinsically kinase-inactive EPHB6 receptor predisposes cancer cells to DR5-induced apoptosis by promoting mitochondrial fragmentation.

Death Receptor 5 (DR5) is a promising target for cancer therapy due to its ability to selectively induce apoptosis in cancer cells. However, the therapeutic usefulness of DR5 agonists is currently limited by the frequent resistance of malignant tumours to its activation. The identification of molecular mechanisms that determine outcomes of DR5 action is therefore crucial for improving the efficiency of DR5-activating reagents in cancer treatment. Here, we provide evidence that an intrinsically kinase-inactive member of the Eph group of receptor tyrosine kinases, EPHB6, induces marked fragmentation of the mitochondrial network in breast cancer cells of triple-negative origin, lacking expression of the estrogen, progesterone and HER2 receptors. Remarkably, this response renders cancer cells more susceptible to DR5-mediated apoptosis. EPHB6 action in mitochondrial fragmentation proved to depend on its ability to activate the ERK-DRP1 pathway, which increases the frequency of organelle fission. Moreover, DRP1 activity is also essential to the EPHB6-mediated pro-apoptotic response that we observe in the context of DR5 activation. These findings provide the first description of a member of the receptor tyrosine kinase family capable of producing a pro-apoptotic effect through the activation of ERK-DRP1 signaling and subsequent mitochondrial fragmentation. Our observations are of potential practical importance, as they imply that DR5-activating therapeutic approaches should be applied in a more personalized manner to primarily treat EPHB6-expressing tumours. Finally, our findings also suggest that the EPHB6 receptor itself may represent a promising target for cancer therapy, since EPHB6 and DR5 co-activation should support more efficient elimination of cancer cells.

In pursuit of synergy: An investigation of the PI3K/mTOR/MEK co-targeted inhibition strategy in NSCLC.

Clinical PI3K inhibition has been somewhat disappointing, due to both inadequate patient stratification and compensatory cell signalling through bypass mechanisms. As such, investigation of PI3K-MEK co-targeted inhibition has been recommended. With high mortality rates and a clear need for new therapeutic intervention strategies, non-small cell lung cancer (NSCLC) is an important setting to investigate the effectiveness of this approach.Here, 174 NSCLC tumours were screened for 150 mutations by Fluidigm technology, with 15 patients being profiled for phosphoprotein expression. The effects of GDC-0941 (a pan PI3K inhibitor), GDC-0980 (a dual PI3K/mTOR inhibitor) and GDC-0973 (a MEK inhibitor) alone and in combination were assessed in 3 NSCLC cell lines.PIK3CA was mutated in 5.17% of NSCLC patients. GDC-0941 and GDC-0980 treatment induced anti-proliferative and pro-apoptotic responses across all NSCLC cell lines, while GDC-0973 treatment induced only anti-proliferative responses. GDC-0980 and GDC-0973 combined treatment led to significant increases in apoptosis and synergistic reductions in proliferation across the panel of cell lines.This study found that the PI3K/MEK co-targeted inhibition strategy is synergistic in all 3 molecular subtypes of NSCLC investigated. Consequently, we would advocate clinical trials for NSCLC patients combining GDC-0980 and GDC-0973, each of which are separately under clinical investigation currently.

Carfilzomib Triggers Cell Death in Chronic Lymphocytic Leukemia by Inducing Proapoptotic and Endoplasmic Reticulum Stress Responses.

Carfilzomib, while active in B-cell neoplasms, displayed heterogeneous response in chronic lymphocytic leukemia (CLL) samples from patients and showed interpatient variability to carfilzomib-induced cell death. To understand this variability and predict patients who would respond to carfilzomib, we investigated the mechanism by which carfilzomib induces CLL cell death. Using CLL patient samples and cell lines, complementary knockdown and knockout cells, and carfilzomib-resistant cell lines, we evaluated changes in intracellular networks to identify molecules responsible for carfilzomib's cytotoxic activity. Lysates from carfilzomib-treated cells were immunoblotted for molecules involved in ubiquitin, apoptotic, and endoplasmic reticulum (ER) stress response pathways and results correlated with carfilzomib cytotoxic activity. Coimmunoprecipitation and pull-down assays were performed to identify complex interactions among MCL-1, Noxa, and Bak. Carfilzomib triggered ER stress and activation of both the intrinsic and extrinsic apoptotic pathways through alteration of the ubiquitin proteasome pathway. Consequently, the transcription factor CCAAT/enhancer-binding protein homology protein (CHOP) accumulated in response to carfilzomib, and CHOP depletion conferred protection against cytotoxicity. Carfilzomib also induced accumulation of MCL-1 and Noxa, whereby MCL-1 preferentially formed a complex with Noxa and consequently relieved MCL-1's protective effect on sequestering Bak. Accordingly, depletion of Noxa or both Bak and Bax conferred protection against carfilzomib-induced cell death. Collectively, carfilzomib induced ER stress culminating in activation of intrinsic and extrinsic caspase pathways, and we identified the CHOP protein level as a biomarker that could predict sensitivity to carfilzomib in CLL. Clin Cancer Res; 22(18); 4712-26. ©2016 AACR.

The NF-κB pathway participates in the response to sulfide stress in Urechis unicinctus

The NF-κB pathway is known to be involved in regulating apoptosis, inflammation and immunity in organisms. In this study, we first identified full-length cDNA sequences of two key molecules in the NF-κB pathway, namely, NEMO and p65, and characterized their responses in the hindgut of Urechis unicinctus (Echiura, Urechidae) exposed to sulfide. The full-length of cDNA was 2491 bp for U. unicinctus NEMO (UuNEMO) and 1971 bp for U. unicinctus p65 (Uup65), and both polyclonal antibodies were prepared using UuNEMO or Uup65 expressed prokaryotically with the sequence of their whole open reading frame. Immunoprecipitation and Western blotting showed that the NF-κB pathway was activated in U. unicinctus exposed to sulfide, in which the content of UuNEMO ubiquitination and nuclear Uup65 increased significantly (p < 0.05) in hindgut tissue of U. unicinctus exposed to sulfide. Furthermore, the mRNA level of UuBcl-xL, a downstream anti-apoptosis gene of the NF-κB pathway, increased significantly (p < 0.05) from 48 h to 72 h and the mRNA level of UuBax, a Bcl-xL antagonist gene, decreased significantly (p < 0.05) at 48 h in the hindgut of U. unicinctus exposed to 50 μM sulfide. During the 150 μM sulfide exposure, the level of UuBcl-xL showed no obvious change, whereas the UuBax mRNA level increased significantly (p < 0.05) at 72 h post-exposure to 150 μM sulfide. We suggested that the activated NF-κB pathway up-regulates UuBcl-xL expression, and evokes an anti-apoptotic response to resist sulfide damage at 50 μM in U. unicinctus. Meanwhile, a Bax-mediated pro-apoptotic response occurs when U. unicinctus is exposed to 150 μM sulfide.

CerS6 Is a Novel Transcriptional Target of p53 Protein Activated by Non-genotoxic Stress

Our previous study suggested that ceramide synthase 6 (CerS6), an enzyme in sphingolipid biosynthesis, is regulated by p53: CerS6 was elevated in several cell lines in response to transient expression of p53 or in response to folate stress, which is known to activate p53. It was not clear, however, whether CerS6 gene is a direct transcriptional target of p53 or whether this was an indirect effect through additional regulatory factors. In the present study, we have shown that the CerS6 promoter is activated by p53 in luciferase assays, whereas transcriptionally inactive R175H p53 mutant failed to induce the luciferase expression from this promoter. In vitro immunoprecipitation assays and gel shift analyses have further demonstrated that purified p53 binds within the CerS6 promoter sequence spanning 91 bp upstream and 60 bp downstream of the transcription start site. The Promo 3.0.2 online tool for the prediction of transcription factor binding sites indicated the presence of numerous putative non-canonical p53 binding motifs in the CerS6 promoter. Luciferase assays and gel shift analysis have identified a single motif upstream of the transcription start as a key p53 response element. Treatment of cells with Nutlin-3 or low concentrations of actinomycin D resulted in a strong elevation of CerS6 mRNA and protein, thus demonstrating that CerS6 is a component of the non-genotoxic p53-dependent cellular stress response. This study has shown that by direct transcriptional activation of CerS6, p53 can regulate specific ceramide biosynthesis, which contributes to the pro-apoptotic cellular response.

Abstract 4710: BRD4 degradation by PROTACs represents a more effective therapeutic strategy than BRD4 inhibitors in ovarian cancer

Abstract BRD4, a member of the bromodomain and extraterminal domain (BET) family of chromatin readers, localizes to super-enhancer regions of DNA, and turns on the expression of a number of oncogenes. Therefore, it has attracted considerable interest as a potential drug target in a variety of cancers, and several inhibitors that block its binding to acetylated histones have been discovered. The BRD4 genomic region is amplified in approximately 17% of ovarian serous adenocarcinomas, and a recent shRNA screen identified it as a bona fide target in high-grade serous ovarian carcinoma (HGSOC). BRD4 inhibitors were shown in the same study to be efficacious in cell culture and PDX models of HGSOC. However, the inhibitor paradigm of drug discovery has several limitations, for example, the requirement of high target occupancy for activity, and the inability to block potentially critical scaffolding functions of the drug target. To circumvent these limitations, we have designed small molecule BRD4 degraders based on our PROTAC (PROteolysis TArgeting Chimera) platform, and assessed their activity across a panel of ovarian carcinoma cell lines. BRD4 PROTACs, containing a BRD4 ligand connected to a VHL E3 ubiquitin ligase binding moiety, successfully promote proteasome-mediated degradation of BRD4 at sub- to low-nM concentrations in a number of ovarian cancer cell lines. Furthermore, this BRD4 degradation results in a very potent anti-proliferative effect in the cells. All the cell lines tested showed modest anti-proliferative effects when treated with BRD4 inhibitors (ED50 ∼ 0.5 μM-1μM). In contrast, when treated with BRD4 PROTACs the cell lines separated into three profoundly different groups (most sensitive, moderately sensitive, least sensitive), with a difference of several orders of magnitude (ED50 ∼ 0.03nM-500nM). Thus BRD4 PROTACs are up to 10,000-fold more potent than BRD4 inhibitors in the most sensitive cell lines. Moreover, the ED50 curves closely track with BRD4 degradation, and result in a much more profound apoptotic response than BRD4 inhibition. In addition, we have also shown that the anti-proliferative effect of BRD4 PROTACs is decoupled from their effect on the levels of cMYC. Finally, we have carried out deep RNA sequencing in five cell lines of variable sensitivity to BRD4 PROTACs in order to thoroughly define the molecular subtype of the malignancies represented and have identified gene signatures that correlate with efficacy of BRD4 PROTACs and BRD4 inhibitors in these cells. In summary, PROTACs designed to target BRD4 for proteasomal degradation are highly potent in ovarian cancer cells, and elicit a powerful pro-apoptotic response in the same setting. The cells can be grouped by their level of sensitivity to PROTAC-mediated BRD4 degradation, and carry gene expression biomarkers which we hope will enable a patient selection strategy as BRD4 PROTACs are advanced into the clinic. Citation Format: Kanak Raina, Jing Lu, Yimin Qian, Martha Altieri, Hanqing Dong, Jing Wang, Xin Chen, Andrew Crew, Kevin Coleman, Craig Crews, James Winkler. BRD4 degradation by PROTACs represents a more effective therapeutic strategy than BRD4 inhibitors in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4710.

Calcineurin complex isolated from T-cell acute lymphoblastic leukemia (T-ALL) cells identifies new signaling pathways including mTOR/AKT/S6K whose inhibition synergize with calcineurin inhibition to promote T-ALL cell death.

Calcineurin (Cn) is a calcium activated protein phosphatase involved in many aspects of normal T cell physiology, however the role of Cn and/or its downstream targets in leukemogenesis are still ill-defined. In order to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-cell acute lymphoblastic leukemia (T-ALL) we used tandem affinity chromatography, followed by mass spectrometry to purify novel Cn-interacting partners. We found the Cn-interacting proteins to be part of numerous cellular signaling pathways including eIF2 signaling and mTOR signaling. Coherently, modulation of Cn activity in T-ALL cells determined alterations in the phosphorylation status of key molecules implicated in protein translation such as eIF-2α and ribosomal protein S6. Joint targeting of PI3K-mTOR, eIF-2α and 14-3-3 signaling pathways with Cn unveiled novel synergistic pro-apoptotic drug combinations. Further analysis disclosed that the synergistic interaction between PI3K-mTOR and Cn inhibitors was prevalently due to AKT inhibition. Finally, we showed that the synergistic pro-apoptotic response determined by jointly targeting AKT and Cn pathways was linked to down-modulation of key anti-apoptotic proteins including Mcl-1, Claspin and XIAP. In conclusion, we identify AKT inhibition as a novel promising drug combination to potentiate the pro-apoptotic effects of Cn inhibitors.

The PINK‐Parkin Circuit in Mitochondrial Quality Control

Mutations in E3 ubiquitin ligase Parkin and Ser/Thr protein kinase Pink1 cause autosomally inherited parkinsonism. Genetic studies have shown that Pink1 and Parkin act in the same pathway that regulates mitochondria dynamics, motility, and quality control. Activation of this pathway can be triggered by a variety of cellular stressor signals that cause mitochondrial damage.Here, we present evidence that PINK1‐Parkin has both cytoprotective and pro‐apoptotic functions. PINK1‐Parkin operates as a molecular switch to dictate cell fate decisions in response to different cellular stressors. Cells exposed to severe and irreparable mitochondrial damage agents such as valinomycin can undergo PINK1‐Parkin‐dependent apoptosis. The proapoptotic response elicited by valinomycin is associated with the degradation of Mcl‐1. PINK1 directly phosphorylates Parkin at Ser65 of its Ubl domain and triggers activation of its E3 ligase activity through an autocatalytic mechanism which amplifies its E3 ligase activity towards Mcl‐1. Autocatalytic activation of Parkin bolsters it accumulation on mitochondria and apoptotic response to valinomycin. Our results suggest that PINK1‐Parkin constitutes a damage‐gated molecular switch that governs cellular context‐specific cell fate decisions in response to variable stress stimuli. We developed an analog sensitive PINK1 allele and demonstrated that PINK1 is required for triggering Parkin translocation to mitochondria, but is dispensable for sustaining Parkin accumulation and consequent mitophagy.Support or Funding InformationThis work was supported by grants from a Butcher Award from the University of Colorado and National Institutes of Health CA107098 and GM113141 to Xuedong Liu. The ImageXpress MicroXL was supported by a NCRR grant S10 RR026680 from NIH.

Innate immune signaling through differential RIPK1 expression promote tumor progression in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a devastating disease for which new treatments, such as immunotherapy are needed. Synthetic double-stranded RNAs, which activate toll-like receptor 3 (TLR3), have been used as potent adjuvants in cancer immunotherapy by triggering a proapoptotic response in cancer cells. A better understanding of the mechanism of TLR3-mediated apoptosis and its potential involvement in controlling tumor metastasis could lead to improvements in current treatment. Using paired, autologous primary and metastatic HNSCC cells we previously showed that metastatic, but not primary tumor-derived cells, were unable to activate prosurvival NF-κB in response to p(I):p(C) resulting in an enhanced apoptotic response. Here, we show that transcriptional downregulation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) in metastatic HNSCC cells causes a loss of TLR3-mediated NF-κB signaling, resulting in enhanced apoptosis. Loss of RIPK1 strongly correlates with metastatic disease in a cohort of HNSCC patients. This downregulation of RIPK1 is possibly mediated by enhanced methylation of the RIPK1 promoter in tumor cells and enhances protumorigenic properties such as cell migration. The results described here establish a novel mechanism of TLR3-mediated apoptosis in metastatic cells and may create new opportunities for using double stranded RNA to target metastatic tumor cells.

Abstract P3-06-10: t-Darpp enhances protein kinase A signaling through direct effects on PKA holoenzyme and indirectly influences cell metabolism, proliferation and apoptosis

Abstract Trastuzumab has dramatically improved the adjuvant therapy of Her2-positive breast cancer. However, acquired resistance to trastuzumab frequently occurs. Breast cancer cells in vitro selected for trastuzumab resistance exhibit over-expression of t-Darpp, a truncated form of the 32 kDa dopamine and cAMP-regulated phosphoprotein (Darpp-32). t-Darpp over-expression has previously been demonstrated to confer trastuzumab resistance and to result in increased protein kinase A (PKA) activity and signaling. Phosphorylation of Darpp-32 at threonine 75 has been shown to promote inhibition (not activation) of PKA by a yet unknown mechanism. T75 phosphorylation on t-Darpp is required for its trastuzumab resistance activity but the role of T75 phosphorylation in PKA activation by t-Darpp is not known and the molecular mechanism by which t-Darpp enhances PKA activity has not been reported. We used cells stably over-expressing either wild type t-Darpp or a T75A mutant of t-Darpp. Using proximity ligation assays, we found that t-Darpp binds directly to the RI regulatory subunit of PKA leading to 75% less RI subunit bound to the catalytic subunit of PKA (PKAc), compared to parental cells. In contrast, the t-Darpp-T75A mutant does not bind RI and those cells have only 34% less RI bound to PKAc compared to parental. These results suggest a direct effect of t-Darpp on PKA activation by binding the regulatory subunit RI and thereby preventing RI from inhibiting PKAc. The T75 phosphorylation site appears to be required for this activity. Using enzymatic and caspase activity assays, we also show that cells over-expressing t-Darpp, but not t-Darpp-T75A, have increased proliferation, up to 4-fold more cellular ATP, and diminished apoptotic response to etoposide, relative to parental cell controls. So far, this data suggests a dual mode of t-Darpp action: modulation of cell metabolism (possibly via effects on ATP production) and direct effects on PKA holoenzyme. Ultimately, pro-apoptotic response is diminished. Further studies will determine whether PKA activation by t-Darpp is responsible for, or separately influenced by, the metabolic effects of t-Darpp. Citation Format: Lenz G, Theile D, Kane SE. t-Darpp enhances protein kinase A signaling through direct effects on PKA holoenzyme and indirectly influences cell metabolism, proliferation and apoptosis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-10.

Eosinophil resistance to glucocorticoid-induced apoptosis is mediated by the transcription factor NFIL3.

The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs' effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils' response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that (1) GCs' TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and (2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don't upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils.

Lovastatin protects keratinocytes from DNA damage-related pro-apoptotic stress responses stimulated by anticancer therapeutics

BackgroundOral mucositis (OM) is a relevant adverse effect of anticancer therapy involving ionizing radiation (IR) and doxorubicin (Doxo). Because DNA damage of keratinocytes is causative for the pathogenesis of OM, we aim to identify pharmacological measures for geno- and cytoprotection of keratinocytes. MethodsWe investigated the influence of the lipid-lowering drug lovastatin on cell death, proliferation and DNA damage response (DDR) mechanisms of human keratinocytes following treatment with IR and Doxo. ResultsLovastatin protected keratinocytes from the cytotoxic and genotoxic effects of IR and Doxo as shown by a diminished induction of apoptosis as well as a reduced formation and slightly improved repair of DNA damage following Doxo and IR treatment, respectively. Lovastatin selectively blocked the activation of Chk1 and ATR kinases following treatment with IR, Doxo and the ribonucleotide reductase inhibitor hydroxyurea, indicating that the statin antagonizes ATR/Chk1-regulated replicative stress responses. Part of the cytoprotective activity of lovastatin seems to rest on a delayed entry of lovastatin treated cells into S-phase. Yet, because the statin also protected non-proliferating keratinocytes from IR- and Doxo-induced cytotoxicity, cell cycle independent protective mechanisms are involved, too. ConclusionsLovastatin attenuates pro-toxic DNA damage-related responses of keratinocytes stimulated by OM-inducing anticancer therapeutics. The data encourage forthcoming in vivo and clinical studies addressing the usefulness of statins in the prevention of OM.

Abstract B31: EZH2 suppresses the interferon-stimulated gene response in non-Hodgkin lymphoma

Abstract Pharmacological inhibition of the histone lysine methyltransferase EZH2 has emerged as a therapeutic strategy for the treatment of non-Hodgkin Lymphoma (NHL), in particular for cases with mono-allelic mutations in the EZH2 catalytic domain. Potent, selective EZH2 small molecule inhibitors have achieved tumor regression in mutant EZH2-containing preclinical lymphoma models and several of these inhibitors are currently engaged in cancer-focused clinical trials. Here, we show that the presence of EZH2 mutations do not always confer EZH2 inhibitor sensitivity. We discovered that EZH2 is usurped by lymphoma cells to attenuate JAK/STAT signaling and to repress pro-apoptotic interferon response genes. EZH2 inhibition results in the broad induction of interferon-stimulated genes (ISGs) in several phenotypically sensitive NHL cell models. In mutant EZH2-containing insensitive models, EZH2 inhibitors synergize specifically with type I interferons (IFNs) in vitro and in vivo to induce ISGs and apoptosis. The profound combinatorial activity of EZH2 inhibitors and type I IFNs is not restricted to NHL models with mutant EZH2 and, is preferentially observed in models that are not affected by either single agent. Molecular consequences of EZH2 inhibitor and type I IFN combinatorial treatment include STAT1 activation, binding of STAT transcription factor complexes to ISGs, reduction of H3K27me3 levels and increase of H3K4me3 levels at ISG transcriptional start sites, and ISG transcriptional activation. The molecular and anti-proliferative effects of the combination can be suppressed by inactivation of the JAK/STAT signaling pathway, by using the JAK inhibitor ruxolitinib. We suggest that EZH2 inhibitors can be therapeutically combined with type I IFNs for the treatment of NHL. Citation Format: William D. Bradley, Chih-Chi Yuan, Feng Zhao, Christina Lee, Kaylyn E. Williamson, Shilpi Arora, Charlie Hatton, Barbara M. Bryant, Patrick Trojer. EZH2 suppresses the interferon-stimulated gene response in non-Hodgkin lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B31.